1. Field of the Invention
The invention generally relates to diagnostic and monitoring methods and assays for cancer and kits that may be used in such methods. More particularly, the application relates to the use of activated Stat5 for diagnosing and monitoring cancer and predicting the prognosis of (breast) cancer patients and the outcome of cancer therapies, especially breast cancer. The invention also relates to screening assays for discovering compounds that affect levels of activated Stat5.
2. Related Art
One of the most pressing health issues today is diagnosing, monitoring and treating cancer and particularly breast cancer. Breast cancer is the leading form of cancer in women, and the second leading cause after lung cancer of cancer death among this population in the United States. In the industrialized world, about one woman in every nine can expect to develop breast cancer in her lifetime. In the United States, the annual incidence breast cancer is about 180,000 new cases and approximately 48,000 deaths each year (Parkin 1998; Apantaku 2000). Approximately two million women living in the United States alone have been diagnosed with breast cancer at some point in their lives. Breast cancer also occurs among men, though far more rarely (approximately 1,600 new cases diagnosed in the U.S. 1998). Treatment for male breast cancer is guided by our understanding of the disease in women.
Despite ongoing improvements in understanding the disease, breast cancer has remained to a large extent resistant to medical intervention. Most clinical initiatives are focused on early diagnosis, followed by conventional forms of intervention, particularly surgery, radiation, hormone suppression, and chemotherapy. Such interventions are of limited success, particularly in patients where the tumor has undergone metastasis. In patients with breast cancer without detectable lymph node metastases, socalled node negative breast cancer, the risk of death from breast cancer recurrence within 10 years is also high, approximately 30% (McGuire, Tandon et al. 1992). There is a pressing need to improve the arsenal of diagnostic tools and methods available to provide more precise and more effective information that will allow successful treatment in the least invasive way possible. Specifically, markers that can identify patients with very low risk of disease recurrence and death after initial surgery would reduce the extent of overtreatment with expensive and potentially toxic supplementary regimes. The invention meets that need by providing new methods and markers for monitoring breast cancer.
Breast Cancer
Development of cancer is a multistep process of genetic alterations that transform normal cells into highly malignant derivatives (Kinzler and Vogelstein 1996; Lengauer, Kinzler et al. 1998). Tumors within the breast may arise from any of its component tissues (e.g. connective tissue and epithelial structures). However, it is the epithelial tissue compartment that gives rise to most common malignant breast neoplasms.
A number of risk factors for carcinoma of the breast have been identified. These include: geographic influences, genetic predisposition, increasing age, length of reproductive life, parity, age at birth of first child, obesity, exogenous estrogens, fibrocystic changes with a typical epithelial hyperplasia and carcinoma of the contralateral breast or endometrium (Cole 1980; Stoll 1998). The chief forms of carcinoma of the breast are classified as infiltrating or noninfiltrating arising in the ducts. These include intraductal carcinoma, comedocarcinoma, simple or usual type (including scirrhous carcinoma), medullary carcinoma, colloid carcinoma, Paget's disease of the breast and tubular carcinoma. Infiltrating and noninfiltrating carcinomas also arise in the lobules and are referred to as in situ lobular carcinoma and infiltrating lobular carcinoma (Simmons and Osborne 1999; Styblo and Wood 1999).
Among the large group of breast cancer patients with localized tumors and without detectable metastases to nearby lymph nodes, many will be cured by surgery because the tumors have not spread to surrounding tissues and lymph nodes. However, others have occult micrometastatic disease and could benefit from supplementary radiation or adjuvant anti-hormone therapy or chemotherapy. There is a need for diagnostic markers to discriminate between tumors with low risk for micrometastatic spread and those with higher risk. Tumor markers that signify low risk of micrometastatic disease may directly affect the therapeutic decision of whether to use supplementary radiation or adjuvant hormone or chemotherapy. Furthermore, such tumor markers may also affect the surgeon's recommendation of whether to choose breast conserving surgery or mastectomy.
The molecular basis of cancer is still being determined. Underlying genome instability facilitates progressive accumulation of growth-promoting traits in premalignant cells under selective pressure from various growth barriers (Cahill et al 1999). Growth-promoting characteristics of cancer include self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis (Hanahan and Weinberg 2000). Associated with this stepwise progression of tumor cells toward increasing malignancy is a gradual loss of tissue-specific cell differentiation.
Loss of tumor cell differentiation appears to be particularly prominent at the transition from localized, surgically curable cancer to metastatic disease (Hart and Easty 1991; Freije, MacDonald et al. 1998; Rivadeneira, Simmons et al. 2000). This transition also is the single most critical determinant of prognosis for patients with solid tumors (McGuire 1991; Tubiana 1999). Assessment of the activity of transcriptional regulators that maintain cell and tissue-specific differentiation in primary tumors may therefore be useful for predicting the risk of occult micrometastases and tumor recurrence. Such informative tumor markers may directly influence treatment decisions by either providing prognostic distinction between low- and high-risk malignancies, or by predicting tumor response to specific adjuvant therapies or tumor response to specific modes of surgery (breast conservation surgery vs. mastetomy).
In breast cancer, receptors for estrogen and progesterone are related to the state of mammary epithelial cell differentiation and have prognostic value for disease outcome in certain cases. Estrogen and progesterone receptor (ER/PR) status is particularly useful as a predictive marker of positive response to adjuvant anti-estrogen therapy in node-positive breast cancer. However, the ER/PR status is not clinically useful to predict prognosis in node-negative cancer (Fitzgibbons, Page et al. 2000). This may be due to the high proportion of ER/PR positive, localized tumors. There is a need to identify low-risk breast cancer patients who may be spared from costly and potentially toxic adjuvant antiestrogen treatment or chemotherapy. There is also a need to identify low-risk breast cancer patients who may benefit from less invasive procedures such as breast conserving surgery, or lumpectomy, with or without post-surgical radiation therapy, instead of mastectomy. The benefits of less extensive and less invasive therapeutic regimes to patients with good prognosis may include avoidance of side-effects, improved mental and physical health, improved quality of life, and lower financial burden. The benefits to society are particularly the cost-saving aspects of avoiding unneccessary overtreatment. One means of accomplishing this is to obtain better prognostic markers for node-negative, as well as other types of breast cancer. These needs are met by the invention.
Diagnosis of Breast Cancer
The definitive diagnosis of all types of breast disease is based on histologic evaluation of tissue samples using the light microscope. The histologic criteria used to define most breast lesions are historic but nonetheless quite reproducible for identifying fully invasive breast cancers.
Improved detection and screening routines, and the development and increasing utilization of fine needle aspirates (FNAs) and core needle biopsies for obtaining tissue samples have been major advances in both detection and diagnosis. Stereotactic image guidance of needle biopsies has tremendously improved our ability to sample suspicious lesions, particularly non-palpable masses, as small as a few millimeters in diameter nearly anywhere in the breast. This has dramatically increased the detection of small, more treatable breast cancers and decreased unnecessary surgery in an enormous number of patients with insignificant benign disease. Recent accomplishments include the identification of a small number of tissue-based biomarkers that are helpful in predicting clinical outcome and response to therapy (e.g., S-phase fraction, estrogen and progesterone receptors, c-erbB-2) and the discovery of genes (BRCA-1 and BRCA-2) associated with familial risk for breast cancers (Dahiya and Deng 1998; Fitzgibbons, Page et al. 2000).
However, diagnosing breast cancer still requires some type of biopsy procedure. In addition, current diagnostic and prognostic methods cannot absolutely distinguish breast cancers that are treatable by surgery alone from those that are likely to recur or have already spread through micrometastases. As a result, at least 50 percent of breast cancer patients with node negative disease are treated with some form of adjuvant therapy. Moreover, available methods are inadequate for predicting the response of breast cancers to specific types of adjuvant therapies.
Treatment decisions for individual breast cancer patients are frequently based on the number of axillary lymph nodes involved with disease, estrogen receptor and progesterone receptor status, size of the primary tumor, and stage of disease at diagnosis (Tandon, Clark et al. 1989). However, even with this variety of factors, it is currently not possible to predict accurately the course of disease for all breast cancer patients. There is clearly a need to identify new markers in order to separate patients with good prognosis, who might need no supplementary therapy beyond surgical removal of the malignant breast tumor, from those whose cancer is more likely to recur and who might benefit from additional and more exhaustive treatment forms.
Despite extensive efforts over several decades, only a limited number of immunohistochemical breast tumor markers have been identified. Among immunohistochemical markers, hormone receptor status remains the only to have gained standard clinical use for evaluating node-negative breast tumors (Fitzgibbons, Page et al. 2000). With improving methods for screening and detection of early breast cancer the proportion of node-negative cases is expected to continue to rise (Elledge and McGuire 1993). Parameters that have been established to be important for the prognosis of patients with breast malignancies in general and that are used by clinicians include: size of primary tumor, stage of disease at diagnosis, number of axillary lymph nodes involved with disease, and hormonal receptor status (ER/PR) (Fitzgibbons, Page et al. 2000). Abnormal status of ErbB-2 or p53, as well as other histological and genetic markers, also are associated with poor prognosis especially in node-positive tumors (Slamon, Clark et al. 1987; Fresno, Molina et al. 1997; Pharoah, Day et al. 1999).
In this regard, U.S. Pat. No. 5,599,681 has suggested the use of an antibody that specifically binds to a reversible phosphorylation site of the c-erbB2 oncoprotein in its active form to screen for the metastatic potential of tumors in patients with node-negative breast cancer. Nowhere, however, was it suggested that screening for activated Stat5 could be used to predict the metastatic potential of breast cancer.
There remain deficiencies in the art with respect to the identification of markers linked with the progression of breast cancer, the development of diagnostic methods to monitor disease progression and the development of therapeutic methods and compositions; to treat breast diseases and cancers. The identification of markers which are differentially expressed or activated in breast cancer would be of considerable importance in the development of a rapid, inexpensive method to improve diagnosing of breast cancer and to predict tumor behavior with respect to patient prognosis and responsiveness to individual therapeutic options. The identified marker(s) would also be useful as a target of therapeutic compositions, of in screening assays for therapeutic compounds.
The diagnostic and monitoring methods of the invention meet many needs in this area.
Therapeutic Regimes for Treating Breast Cancer
Treatment of breast cancer is multifaceted and complex. The choice of therapeutic approach is guided by a series of criteria based on a limited set of tumor characteristics. Nearly all patients with breast cancer will have some type of surgery. This may be supplemented by local therapy with radiation, or by systemic therapy including hormone suppression or chemotherapy. To kill cancer cells that may have spread beyond the breast and nearby tissues, physicians employ oral or intravenous systemic therapy. Examples of systemic treatments for breast cancer are chemotherapy and antiestrogen therapy. Systemic therapy given to patients after surgery is often referred to as adjuvant therapy. The goal of adjuvant therapy is to kill hidden cancer cells. Even in the early stages of the disease cancer cells can break away from the primary breast tumor and spread through the bloodstream. These cells usually cause no detectable symptoms and usually do not show up on an x-ray and cannot be felt during a physical examination. But they can establish new tumors in other locations in the body. Furthermore, oncologists sometimes give patients neo-adjuvant therapy—that is, systemic therapy before surgery, typically to shrink the tumor.
The following summarizes the main principles of treatment of breast cancer according to current guidelines endorsed by the U.S. National Cancer Consortium Network and the American Cancer Society (1999). The text below maintains an emphasis on treatment of node-negative breast cancer, as it relates to the present invention.
Breast conserving surgery—“Lumpectomy” removes only the breast lump and the surrounding area, or margin, of normal tissue. If cancer cells are present at the margin (the edge of the excisional biopsy or lumpectomy specimen), a re-excision can usually be done to remove the remaining cancer. In most cases, lumpectomy is combined with 6 to 7 weeks of supplementary radiation therapy following surgery. This combination of lumpectomy and radiation is often referred to as “breast conserving therapy”.
Mastectomy—In a “simple (total) mastectomy” procedure surgeons remove the entire breast but do not remove any lymph nodes from under the arm, or muscle tissue from beneath the breast. In a “modified radical mastectomy”, surgeons remove the entire breast and some of the axillary (underarm) lymph nodes. Modified radical mastectomy is the most common surgery for patients with breast cancer in whom doctors remove the whole breast. “Radical mastectomy” removes not only the entire breast, but axillary lymph nodes and the chest wall muscles under the breast as well. The less extensive modified radical mastectomy has proved as effective as radical mastectomy, which is nowadays rarely performed due to disfiguration and frequent side-effects.
Lymph node surgery—Regardless of whether a breast cancer patient has a mastectomy, or a lumpectomy for invasive cancer, the physicians need to determine whether the cancer has spread. The regional lymph nodes in the underarm drain lymph from the breast, and are typically the first sites of spread. Furthermore, lymph node involvement increases the likelihood that cancer cells have spread through the blood-stream to other parts of the body.
While lymph node surgery itself does not improve the chance for a cure, this is the only way to accurately determine if the cancer has spread to the lymph nodes. This usually means removing some or all of the lymph nodes in the armpit. Typically 10 to 20 lymph nodes in the armpit are examined by an operation called “axillary lymph node dissection”. Although axillary lymph node dissection is a safe procedure with low rates of serious side effects, efforts are ongoing to develop new ways of detecting the spread of cancer to lymph nodes that are less invasive and do not involve a full lymph node dissection. Such alternative methods include the “sentinel lymph node biopsy” (Orr, Hoehn et al. 1999; Sugg, Ferguson et al. 2000), and new detection methods for breast cancer cells in bone marrow and blood (Berois, Varangot et al. 2000; Braun, Pantel et al. 2000; Fetsch, Cowan et al. 2000; Ikeda, Miyoshi et al. 2000; Kraeft, Sutherland et al. 2000; Zhong, Kaul et al. 2000). It is possible that these newer methods in the future may replace lymph node dissection as a means of determining micrometastatic spread of cancer.
Sentinel lymph node biopsy—In the sentinel lymph node biopsy procedure the surgeon finds and removes the ‘sentinel node’—the first lymph node into which a tumor drains, and therefore the one most likely to contain cancer cells. Many doctors recommend it for most women with breast cancer, but others still consider it investigational. In a sentinel lymph node biopsy the surgeon injects a radioactive substance and/or a blue dye into the area around the tumor. Lymphatic vessels carry these materials into the sentinel node. The doctor can either see the blue dye or detect the radioactivity with a geiger counter, and then cuts out the node for examination. If the sentinel node contains cancer, the surgeon will have to perform an axillary dissection—removal of more lymph nodes in the axilla (armpit). If the sentinel node is cancer-free, the patient and her physicians may consider avoiding more lymph node surgery and its potential side effects. Although the sentinel node procedure is relatively new and its long-term effectiveness is uncertain (Orr, Hoehn et al. 1999; Sugg, Ferguson et al. 2000), it may turn out to be equally as effective in determining lymph node spread as full lymph node dissection.
Detection of disseminated cancer cells in blood and bone marrow—Recent methods for detecting metastatic breast cancer cells in blood (Berois, Varangot et al. 2000; Fetsch, Cowan et al. 2000; Kraeft, Sutherland et al 2000) or in bone marrow (Braun, Pantel et al. 2000; Ikeda, Miyoshi et al. 2000; Zhong, Kaul et al. 2000) are typically based on the detection of cytokeratin markers characteristic to breast cancer cells by immunological methods or by gene-based testing. These new methods may also lead to an alternative approach to lymph node dissection for determining whether a breast cancer has spread beyond the local tumor area.
Radiation therapy—Radiation is used to destroy cancer cells left behind in the breast, chest wall, or lymph nodes after surgery. Radiation treatments usually take place 5 days a week over a period of 6 to 8 weeks. Side effects most likely to occur include swelling and heaviness in the breast, sunburn-like skin changes in the treated area, and fatigue. Changes to the breast tissue and skin usually go away in 6 to 12 months. In some women, the breast becomes smaller and firmer after radiation therapy. Radiation therapy of axillary (armpit area) lymph nodes can also cause lymphedema. Although generally safe, it is evident that radiation therapy comes at a considerable expense and with potentially serious side-effects. Radiation therapy also involves a major risk for abnormal fetal development, and cannot be used to treat pregnant women with breast cancer.
New tumor markers that signify good prognosis may reduce the need for supplementary radiation therapy.
Chemotherapy—Patients receive this treatment of anti-cancer drugs intravenously (injected into a vein) or by mouth. Either way, the drugs travel in the bloodstream and move throughout the entire body. Doctors who prescribe these drugs (medical oncologists) generally use a combination of medicines proven more effective than a single drug. For women with node-negative breast cancer the most frequently used chemotherapy options are CMF (cyclophosphamide, methotrexate, and fluorouracil), CAF (cyclophosphamide, doxorubicin), and AC (doxorubicin (Adriamycin) and cyclophosphamide) (1999). Doctors give chemotherapy in cycles, with each period of treatment followed by a recovery period. The total course of chemotherapy usually lasts 3 to 6 months depending on the combinations used. This is significant both in terms of cost and reduced well-being. The side effects of chemotherapy are many and potentially severe, and depend on the type of drugs used, the amount taken, and the length of treatment. Doxorubicin and epirubicin may cause heart damage, although doctors limit the dose and perform periodic tests to check heart function in order to prevent this side effect. Other side effects include loss of appetite, nausea and vomiting, mouth sores, hair loss, and changes in the menstrual cycle. Because chemotherapy can damage the blood-producing cells of the bone marrow, a drop in white blood cells can raise a patient's risk of infection, a shortage of blood platelets can cause bleeding or bruising after minor cuts or injuries; and a decline in red blood cells can lead to fatigue due to anemia.
New tumor markers that identify patients with excellent prognosis may eliminate the need for adjuvant chemotherapy among these patients.
Hormone therapy—Estrogen, a female sex hormone produced by the ovaries, promotes growth of some breast cancers. Doctors use several approaches to block the effect of estrogen or to lower estrogen levels. The most commonly used antiestrogen drug is tamoxifen, taken daily in pill form, usually for 5 years. Studies show that tamoxifen can reduce the chances of breast cancer coming back after surgery if the breast cancer cells contain receptors for estrogen or progesterone. Tamoxifen may be used to treat metastatic breast cancer, but also a significant number of patients with node-negative cancer receive tamoxifen treatment.
Adjuvant Herceptin therapy—A new form of adjuvant breast cancer treatment involves the use of Herceptin, a drug that antagonizes activity of the Her2/neu oncogene reecently introduced for select patients with node-positive breast cancer (Stebbing, Copson et al. 2000). Herceptin therapy will not be discussed in more detail here.
Therapeutic considerations in node-negative breast cancer—Decisions about types of surgery (breast conserving lumpectomy, radical mastectomy), radiation therapy, adjuvant chemotherapy or hormonal therapy are currently based on the status of axillary lymph nodes, the size of the malignant tumor and its histologic type (appearance under a microscope), and hormone receptor status. For example, if regional lymph nodes are negative (do not contain any cancer cells) and the tumor measures half a centimeter or smaller, the patient needs no adjuvant (post-surgery) therapy. In current practice, a substantial number of patients with node-negative breast cancer with larger tumors receive adjuvant therapies with questionable benefit in terms of relatively limited improvement in prognosis considering the associated increased morbidity and serious side-effects (McGuire, Tandon et al. 1992). These adjuvant therapies also come at high cost as described above. Furthermore, the choice of the less invasive breast conserving surgery (lumpectomy) is generally preferred by doctors and patients over mastectomy, but more specific guidelines and better prognostic tumor markers are needed to guide this selection. There is therefore a strong need for new markers to identify breast cancer patients with low risk for disease recurrence and death.
Markers for low-risk cancer and patient follow-up—Better prognostic tumor markers may also have the benefit of reducing the frequency of follow-up visits among patients with low-risk cancer. Tumor markers identifying low-risk breast cancer patients may also allow reduced frequency and lighten the extensive requirements for patient follow-up. While this is primarily a cost issue, it also positively impacts the patient's quality of life. Routine surveillance and follow-up for all patients who have had invasive breast cancer curently includes the following: a history and physical exam every 4-6 months for 2 years, then every 6 months for 3 years, and then, once every year (1999). Women who have had a lumpectomy and radiation (breast conservation therapy) should undergo mammography of the treated breast at 6 months after radiation therapy, and then mammography of both breasts on an annual basis. Women who have had a mastectomy should get a mammogram of the remaining breast annually after the surgery. Because tamoxifen increases a postmenopausal woman's risk of developing cancer of the endometrium (lining of the upper part of the uterus), postmenopausal patients taking this drug also should have an annual pelvic exam. Markers indicating low-risk for tumor recurrence therefor may benefit both patients and society by reduced costs associated with fewer and less extensive follow-up examinations.
Monitoring of recurrent breast cancer—Work-up for a suspected recurrence of breast cancer includes a biopsy to confirm the first recurrence whenever possible. A recurrence may be local, meaning that cancer has returned to the breast, underarm lymph nodes, or nearby tissues, or systemic, which means that cancer has spread to distant organs. There exist a series of guidelines to treat locally recurring breast cancer. The current recommendations for treatment of the locally recurring tumor depend in large part on what mode of treatment was used for the original tumor (1999). New markers that predict the biological behavior of breast cancer may affect the choice of follow-up therapy, depending on whether the recurrent tumor is deemed low or high risk. For instance, local recurrence of a tumor positive for a marker indicating low risk of distant spread may allow the use of less intensive therapeutic approaches than if the tumor is negative for this same marker. For example, reexcision and possibly local radiation may suffice instead of radical mastectecomy with or without adjuvant chemotherapy or anti-hormone therapy.
Stat5
The Signal Transducer and Activator of Transcription (STAT) family of transcription factors provide a signaling link between cell surface hormone and cytokine receptors and specific response elements in the promoters of selective genes. Seven mammalian STAT genes have been identified. The Stat5 transcription factor is involved in regulation of cell growth, differentiation, and cell survival (Wakao, Gouilleux et al. 1994). It exists as two highly homologous isoforms, Stat5a and 5b, which have more than 95% amino acid homology and are encoded by separate genes (Liu, Robinson et al. 1995; Grimley, Dong et al. 1999). Stat5 is required for normal mammary epithelial cell development and differentiation (Liu, Robinson et al. 1997; Udy, Towers et al. 1997; Moriggl, Topham et al. 1999).
Stat5 polypeptides typically are cytoplasmic and quiescent under homeostatic conditions. Their activation results from phosphorylation of the highly conserved C-terminal tyrosine at Tyr694 in Stat5a or the corresponding Tyr699 in Stat5b by certain intracellular tyrosine kinases. This phosphorylation permits dimer pair formation which is needed for Stat5 to bind to DNA.
This initial phosphotyrosyl “on-switch” is a generic Stat feature (Darnell 1997; Darnell 1998) and is triggered when cells with cognate receptors are exposed to a variety of stimuli including cytokines, immune complexes, microbiologic agents or non-peptidyl compounds. Although the spectrum of agonists thus is heterogeneous, the bulk implicated in triggering Stat5 activation belong to the class I and class II cytokine superfamilies. (See Table 4 of (Grimley, Dong et al. 1999). These cytokines utilize receptors lacking a catalytic domain (Liu, Gaffen et al. 1998), so that the Stat activation is most often dependent upon an auxiliary protein kinase (Leonard and O'Shea 1998).
The Janus tyrosine kinases (Jaks) form biochemically stable associations with class I and class II cytokine receptors. A non-covalent linkage facilitates Jak phosphorylations during receptor ligation and increases the odds of interactions between Jaks and Stat5 recruited to receptor-Jak complexes (Leonard and O'Shea 1998). This critical and conserved mutual relationship has engendered the scientific vernacular of “Jak-Stat pathway” (Liu, Gaffen et al. 1998). However, Jaks are not the sole means of Stat activation.
Stat5a and Stat5b can also be tyrosine phosphorylated by a number of cytokines commonly designated as “growth factors” which bind to receptor tyrosine kinases (RTKs). The RTKs possess intrinsic catalytic properties, and may trigger Stat5 signals absent a direct linkage to the Jak enzyme system (Chen, Sadowski et al. 1997). In addition, Stat5 tyrosine phosphorylation might be effected by cytosolic protein kinases in the Src or Tec families. As “nonreceptor tyrosine kinases” (NTKs), the latter enzymes can function without extrinsic stimulation due to receptor ligation. The Src-family kinase Lck has been implicated in Stat5 phosphorylation during T cell proliferation (Welte, Leitenberg et al. 1999) and constitutively active NTKs, RTKs or analogous oncoproteins may be particularly significant in maintaining a constitutive phosphorylation of Stat5 in autonomously proliferating neoplastic cells (For example, See (Lacronique, Boureux et al. 1997; Wellbrock, Geissinger et al. 1998)).
In addition to the initial activation switch of Stat5, which involves phosphorylation of a tyrosine residue within a conserved C-terminal segment and causes dimerization of Stat5 molecules (Gouilleux, Wakao et al. 1994), a second coordinated activation event is required for functional activation. This involves translocation of dimerized Stat5 from the cytoplasm into the cell nucleus, which permits Stat5 to come in proximity of and bind to gene regulatory promoter elements, and thus regulate transcription of specific genes (Gouilleux, Wakao et al. 1994; Kazansky, Kabotyanski et al. 1999). Because Stat5 not only requires phosphorylation of a specific tyrosine residue, but also needs to translocate into the cell nucleus in order to function as an active DNA-binding transcription factor, amounts of tyrosine phosphorylated Stat5 located within the cell nucleus will reflect the levels of activated Stat5 more accurately than overall cellular levels of tyrosine phosphorylated Stat5. For instance, tyrosine phosphorylation of Stat5a by the Src tyrosine kinase has been shown not to be accompanied by nuclear translocation (Kazansky, Kabotyanski et al. 1999), illustrating that quantitation of tyrosine phosphorylation status alone without assessing nuclear localization is not sufficient for accurate determination of levels of activated Stat5. Correspondingly, Stat transcription factors may become dephosphorylated within the cell nucleus and loose the ability to bind to DNA (Haspel and Darnell 1999), making assays that detect nuclear Stat5 protein levels alone also not sufficient for accurate determination of levels of activated Stat5. In the present description, the term ‘levels of activated Stat5’ refers to levels of tyrosine phosphorylated Stat5 within the cell nucleus.
Antibodies that bind exclusively to tyrosine phosphorylated Stat5 can be used to detect activated Stat5 in the nuclei of cells by immunocytochemistry or immunohistochemistry, provided that proper steps are taken to achieve antigen retrieval of this cryptic antigenic site. This antigenic site is cryptic, or unavailable, unless the phosphorylated tyrosine bound to the SH2 domain of the partner molecule in the dimer is dissociated by specific treatment.
Detection of active, tyrosine phosphorylated Stat5 by immunohistochemistry in tissue sections has been reported (Jones, Welte et al. 1999). Stat5 activation in normal mouse mammary gland tissue in response to Erb-B4 activation was studied. However, human breast tissue or human breast cancer samples were not examined. In further contrast to Jones, the current invention may use a simple one-step antigen-retrieval method for determining levels of activated Stat5.
The extent to which Stat5 promotes cell proliferation or inhibits growth by inducing cell differentiation in various tissues, including mammary gland, is unresolved. The possibility that Stat5 activation status is of prognostic value for breast cancer was not obvious prior to the inventors' discovery, because a priori, it had been argued that Stat5 activation may promote mammary tumor formation instead of being associated with reduced risk of invasion and metastasis.
It was specifically suggested that a general anti-apoptotic effect of Stat5 might contribute to mammary tumor progression in rodents (Humphreys and Hennighausen 2000). This notion was supported by the observation that in mice lacking the Stat5a gene (Stat5a−/− mice) but overexpressing the oncogenic TGF-alpha transgene, the rate of mammary tumor formation was reduced relative to that observed in Stat5a+/+ mice (Humphreys and Hennighausen 1999)). This suggested that the Stat5a transcription factor promotes mammary tumor formation. Likewise, a positive role for Stat5 in mammary carcinogenesis indirectly has been indicated by the reduced mammary tumor formation in mice lacking the gene for prolactin, a major activator of Stat5 in mammary epithelial cells (Vomachka, Pratt et al. 2000), as well as the observation that circulating prolactin levels correlated with increased risk of breast cancer in post-menopausal women (Hankinson, Willett et al. 1999). Furthermore, the notion of a tumorigenic role of Stat5 in the mammary gland (Humphreys and Hennighausen 1999; Humphreys and Hennighausen 2000) would be consistent with the prevailing view of a tumor-promoting role of Stat5 in hematopoietic cancer (lymphomas, leukemias) (Wellbrock, Geissinger et al. 1998; Bromberg and Darnell 2000).
Alternatively, it could be argued that Stat5 activation may suppress breast tumor formation by acting as a growth-inhibitory differentiation factor for mammary epithelial cells. Likewise, Stat5 regulates normal differentiation of ovaries and prostate (Teglund, McKay et al. 1998; Moriggl, Topham et al. 1999; Nevalainen, Ahonen et al. 2000). However, there is currently no direct evidence available demonstrating a role of Stat5 as a tumor suppressor in either breast or other tissues. Therefore, the present invention and description of activated Stat5 as a marker of good prognosis in node-negative human breast cancer was unexpected based on the published literature and prevailing views within the scientific field. As such, the role of Stat5 in human breast cancer development and progression had not been established, and its use as a marker of biologic behavior of human breast tumors had not been reported.